This is a collaborative proposal to develop inhibitors with reduced susceptibility to resistance and improved genotype spectrum of activity against the NS3/4A protease of the Hepatitis C virus (HCV). Over three percent of the world's population is infected with the hepatitis C virus (HCV). Unfortunately, the current best treatment is still quite challenging against the most prevalent genotype 1. The HCV NS3/4A protease is an attractive drug target due to its essential role in viral replication. In prior work, we discovered two novel scaffolds that show inhibitory activity against four of the most prevalent genotypes, one of which shows an IC50 value of ~2 M against wild type NS3 genotype 1b, and also maintained its potency within a 10-fold range against five drug-resistant mutants. Another, with an IC50 of ~2-20 M against the more common genotypes will provide a backup hit. We propose to develop these scaffolds into a low molecular weight NS3/4A inhibitor with broader spectrum activity and fewer side effects than current therapeutics. We will pursue this goal through three targeted aims to (1) utilize scaffold expansion of current hits to develop more extensive Structure Activity Relationships (SARs) to increase potency by at least an order of magnitude; (2) use structure-based design and synthesis to further improve inhibitors; and (3) utilize metabolic stability and related pharmacokinetic parameters, along with HCV antiviral efficacy to iteratively improve inhibitor design therapeutic characteristics. With these Aims, we expect to attain Milestone criteria for success that include: reducing the inhibitor enzymatic IC50 to 10 nM; obtaining antiviral EC50 100 nM (replicon assay); retaining good enzymatic selectivity for NS3/4A vs. other off-target enzymes; mouse microsomal stability, t1/2 > 30 min; potential for good oral bioavailability; and minimal cytotoxicity, with a selectivity index, SI 100.